用于灰茭鉴别的菰黑粉菌SNP位点挖掘及验证  

Mining and Verification of SNP Loci of Ustilago esculenta Used for Grey Jiaobai Identification

作　　者：吴骏成 葛鑫涛 杨梦飞 叶子弘 夏文强 汤近天 俞晓平 张尚法 张雅芬 WU Jun-Cheng;GE Xin-Tao;YANG Meng-Fei;YE Zi-Hong;XIA Wen-Qiang;TANG Jin-Tian;YU Xiao-Ping;ZHANG Shang-Fa;ZHANG Ya-Fen(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine/College of Life Sciences,China Jiliang University,Hangzhou 310018,China;Hangzhou C&Y Pollen Pharmaceutical Co.,Ltd.,Hangzhou 310018,China;Jinghua City Academy of Agricultural Sciences,Jinghua 321017,China)

机构地区：[1]中国计量大学生命科学学院/浙江省生物计量与检验检疫技术重点实验室,杭州310018 [2]杭州仟源保灵药业有限公司,杭州310018 [3]金华市农业科学研究院,金华321017

出　　处：《农业生物技术学报》2023年第2期383-392,共10页Journal of Agricultural Biotechnology

基　　金：浙江省大学生科研创新活动计划资助项目(2020R409031);金华市科技计划项目(2020-2-010);浙江省自然科学基金(LY19C140001)。

摘　　要：茭白作为我国重要的水生蔬菜,田间近20%的灰茭比例对其产量造成了严重影响。研究表明,田间正常茭和灰茭两种表型的产生与菰黑粉菌(Ustilago esculenta)菌株分化密切相关,由T型菰黑粉菌侵染菰(Zizania latifolia)所致。由此,本研究通过重测序技术,经SNP calling和SNP的过滤获得在MT型和T型菰黑粉菌中具有差异性的129 494个SNP位点后,通过哈德-温伯格平衡检测和短片段筛除策略,得到了具有T型菰黑粉菌特异性的SNP位点647个。最后采用随机化原则挑选48个位点,设计特异性引物并用等位基因特异性PCR(allele specific PCR, AS-PCR)鉴定筛选出了其中准确率高的8个位点用于田间测试。结果表明,采用8个特异性位点组合分析茭白种苗的T型菰黑粉菌并剔除灰茭鉴别结果阳性的种苗,可将灰茭比例降低37.59%。此研究为进一步提高良种繁育中种苗纯度及后续种苗质量检测提供了技术支持。Jiaobai as an important aquatic vegetable in China, nearly 20% of the grey Jiaobai in the field has a serious impact on its yield. Previous research showed that the formation of 2 phenotypes of normal Jiaobai and grey Jiaobai in the field was closely related to the differentiation of MT and T type strains of Ustilago esculenta. Grey Jiaobai is caused by the infection of Zizania latifolia by T type strain of U. esculenta.Therefore, 129 494 SNP loci with difference between MT and T-type strains was obtained in this study, by resequencing, SNP calling and SNP filtration. After that, 647 specific SNP loci with specificity of T type of strain were screened by Hardy-Weinberg equilibrium test strategy and short fragment sieving. Finally, based on the 48 loci selected by randomization principle, 8 loci with high accuracy were screened by allele specific PCR(AS-PCR) with designed specific primers for field test. The results showed that the occurrence ratio of grey Jiaobai could be reduced 37.59% after eliminating the seedlings with positive identification results using the combination of the selected 8 specific loci to identify the T-type strain. This research provides technical support for further improving the purity of seedlings in improved seed breeding and subsequent seedling quality detection.

关 键 词：茭白 菰黑粉菌 SNP 等位基因特异性PCR(AS-PCR) 灰茭鉴别 

分 类 号：S338[农业科学—作物遗传育种]